Published online 19 September 2007
Nucleic Acids Research, 2007, Vol. 35, No. 19 6439–6450 doi:10.1093/nar/gkm553
Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture Tanya V. Aspinall1, James M.B. Gordon1, Hayley J. Bennett1, Panagiotis Karahalios1, John-Paul Bukowski1, Scott C. Walker2, David R. Engelke2 and Johanna M. Avis1,* 1
Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK and 2Department of Biological Chemistry, 3200 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, Michigan 48109-0606, USA
Received April 12, 2007; Revised June 29, 2007; Accepted July 6, 2007
ABSTRACT
INTRODUCTION
Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins selfinteract, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with prerRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.
RNase MRP is an essential eukaryotic ribonucleoprotein endonuclease, first identified as having a role in mitochondrial DNA replication (1). Subsequent experiments have shown RNase MRP is primarily localized in the nucleolus (2,3), where it functions in pre-rRNA processing, cleaving at a specific site (A3) in the ITS1 of pre-rRNA, leading to the generation of the mature 50 end of 5.8S rRNA (4,5). More recently, a critical role within the cell cycle has emerged, where RNase MRP promotes degradation of CL2B mRNA in Saccharomyces cerevisiae by cleavage within its 50 UTR (6), possibly in cytoplasmic ‘processing bodies’ (7). In the human RNase MRP, mutations in the RNA component have been shown to cause the genetic disease cartilage hair hypoplasia (8). Eukaryotic RNase MRP is structurally and functionally related to the ubiquitous ribonucleoprotein RNase P, which predominantly functions in the processing of pretRNAs. Recent evidence indicates RNase P also plays a role in pol III transcription of its RNA substrates (9), potentially providing a route for coordination of transcription with processing. In S. cerevisiae, RNase P and RNase MRP consist of 9 and 10 known protein subunits, respectively, and 1 distinct RNA molecule. Eight proteins are subunits of both RNase P and MRP (10), with each complex possessing one or more unique protein subunits; Snm1p and Rmp1p in RNase MRP and Rpr2p in RNase P (10,11). The conserved properties of both complexes suggest that they evolved from a common eukaryotic ancestor, the RNA subunit having diverged and unique protein subunits evolved accordingly (12). Indeed, phylogenetic analysis of the RNA subunits yields highly similar
*To whom correspondence should be addressed. Tel: +44 161 306 4216; Fax: +44 161 306 5201; Email:
[email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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secondary structures despite limited sequence similarity (13–17). In bacteria and some archaeal species, the RNA subunit of RNase P has been shown to cleave pre-tRNA in vitro in the absence of protein subunits (18,19). In S. cerevisiae and other eukaryotes, the protein–RNA ratio is much higher and the proteins appear to be a necessity for efficient enzymatic activity in vitro. Very recent studies on the human and Giardia lamblia P RNAs in the absence of proteins, however, have demonstrated low pre-tRNA RNA cleavage activity (20). Evidence is thus in favour of the RNA subunit providing the catalytic core of eukaryotic RNase P/MRP and the protein subunits having likely evolved to assume roles in RNA subunit folding and stabilization and/or to assist substrate binding and catalysis during one or more of the multiple functions of the respective enzymes. Studies on the overall subunit composition and organization of the RNase MRP and RNase P complexes have proved challenging, primarily due to difficulty in obtaining biochemically purified native complexes and soluble individual purified protein subunits for reconstitution studies. However, yeast two-hybrid and yeast threehybrid analyses of both the human and S. cerevisiae RNase P complexes (21–23), and GST pull-downs using the human RNase MRP subunits (24) have provided insights into mutual subunit interactions, and revealed that numerous protein-protein and RNA–protein interactions probably occur in both complexes (reviewed by Walker and Engelke, 2006 (25)). Exploration of direct subunit interactions through in vitro binding studies is lacking on the yeast RNase P and MRP enzymes. Here, we seek to redress this imbalance and to complement existing yeast two-hybrid data to obtain an improved understanding of the structural organization of yeast RNase MRP. This study reports the first successful soluble expression and purification of all 10 of the RNase MRP protein subunits and identifies oneto-one protein–protein and protein–RNA interactions. Comparative analysis with existing data on the yeast and human RNase P and/or RNase MRP systems (25), together with our novel data on protein-pre–rRNA interactions, provides new discussion of the enzyme architecture and protein subunit roles. MATERIALS AND METHODS Expression and purification of GST-fusion proteins POP1, POP3, POP4, POP5, POP6, POP7, POP8 and RPP1 open reading frames were excised out of previously described p413Gal yeast expression vectors (10) and inserted at the SalI-XmaI sites of pGEX-6P-1 (GE Healthcare). Genes for Snm1p and Rmp1p were amplified from S. cerevisiae genomic DNA and cloned into pGEX6P-1 between EcoR1 or BamH1 and Xho1 sites. A KS/ pGEX construct allowing expression of a fragment of Snm1p (residues P124–S198) representing a lysine/serine (K/S)-rich domain of this protein was also created. All constructs were modified by insertion of a sequence at the BamH1 site that places a phosphorylation
site (RRASV) for bovine heart protein kinase A (Sigma) at the N-terminus of the native protein sequence. The expression constructs were routinely transformed into E. coli BL21 RIL cells and grown with appropriate antibiotic selection. A subsequent overnight culture was diluted 1:200, and grown at 378C to an OD600 = 0.8 at which stage expression was induced overnight at 168C by addition 0.1 mM isopropyl b-D-galactopyranoside (IPTG). Pelleted cells were lysed (lysozyme and freeze-thaw cycles) in 25 mM Tris–Cl, pH 8.0, 1 M NaCl, supplemented with 1% Triton X-100, 10 mM dithiothreitol (DTT) and protease inhibitors (Complete, Roche). GST-fusion proteins were purified by affinity chromatography using glutathione Sepharose 4B, GS4B (GE Healthcare). The GS4B beads were prepared and washed with 1 PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), supplemented with 1% Triton X-100 + 10 mM DTT. The same buffer was used for wash and elution steps after application of GST-fusion protein. A GST-only vector (no insert sequence) was included as a control. The concentration and purity of GST-fusion protein on the beads was visually determined by SDS-PAGE. Radiolabelling of the GST-fusion proteins and cleavage from GST GS4B beads bearing bound fusion protein (bedvolume 1 ml) were equilibrated in HMK buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 12 mM MgCl2) and then incubated with 100 U bovine heart kinase solution and 50 mCi [g-32P] ATP (30 min, 48C). The reaction was terminated using 10 ml of stop solution (10 mM sodium phosphate, pH 8.0, 10 mM sodium pyrophosphate, 10 mM EDTA, 10 mg BSA) and the beads subsequently washed with 5 10 ml of 1 PBS + 1% Triton X-100 + 10 mM DTT. Bead samples were analysed by SDS-PAGE, followed by exposure to PhosphoImager screens. The efficiency and specificity of radiolabel incorporation was determined by autoradiography analysis (Typhoon scanner; AP Biotech). Radiolabelled GST-fusion proteins were subsequently cleaved overnight at 48C with PreScission protease (AP Biotech), following the manufacturers recommended protocol. The purity of the cleaved proteins, estimated by gel analysis (with Coomassie staining), was improved (80% pure on average) over that of their respective GST-fusions, though the yield is reduced due to incomplete cleavage from GST (data not shown). The concentration of cleaved, untagged proteins was determined using the Bio-Rad protein assay. In vitro transcription of RNase MRP RNA and pre-rRNA NME1, the coding sequence for yeast MRP RNA, has been cloned into a pUC based vector, immediately downstream of a T7 promotor, creating the construct pHST7/NME1 for run-off transcription [a version of pHST7NME1 described previously (17), without flanking ribozymes]. A pHST7/RPR1 construct was also generated to enable transcription of yeast P RNA. A region encoding a 187 nt RNA fragment starting 73 nt upstream of the A3 site in yeast pre-rRNA ITS1 (at the A2 site ACAC sequence) and ending 114 nt downstream was
Nucleic Acids Research, 2007, Vol. 35, No. 19 6441
amplified from yeast genomic DNA and cloned into the same pUC-derived plasmid, creating the construct pHST7/pre-rRNA for use as an in vitro transcription template. All plasmid template sequences were confirmed via automated DNA sequencing. RNA was transcribed (378C, 2 h) from EcoRI-linearized plasmid template in 20 ml reactions containing 80 mM Tris–HCl (pH 8.0), 2 mM spermine, 40 mM DTT, 30 mM MgCl2, 40 U RNasin, 7 mM rNTPs, [alpha-32P] UTP, 5U yeast inorganic pyrophosphatase, 15 U T7 RNA polymerase. Radiolabelled UTP was omitted for preparation of unlabelled competitor RNA. After transcription, RNAs were purified by treatment with RNase-free DNase I followed by acid phenol/chloroform extraction and ethanol precipitation and then taken through a refolding procedure (808C for 3 min, cooled to 488C at 28C/min then cooled to 208C at 18C/min) in 30 mM HEPES pH 7.5 with 100 mM KCl (1 mM RNA). Conformational homogeneity of RNAs was monitored by UV absorbance melting profiles and by native 4% PAGE.
aggregates commonly observed in RNA–protein interaction studies and to ensure RNA trapped on the nitrocellulose is part of a low ratio (preferably 1:1 or similar) complex(es). In our studies, at least 50% of the radiolabel is retained on the top filter for all proteins. Similar levels of aggregation are observed if fusion protein is used. The percentage bound values reported exclude RNA aggregates and are a percentage of the soluble RNA bound [counts per minute (c.p.m.) retained on nitrocellulose filter/(total input c.p.m. c.p.m. on 0.45 mm filter]. For those subunits observed to bind MRP RNA in the filter binding screens, relative competition by poly(U), MRP RNA and P RNA was assessed by incubation of 32 P-MRP RNA bound protein complexes with 1–1000 nM unlabelled RNA competitor.
GST pull-down assay
S. cerevisiae RNase MRP comprises one RNA molecule and at least 10 protein subunits, but little is known about the overall architecture of the complex. The approach taken here was to obtain each protein subunit in a recombinant form in order to screen for direct subunit interactions in vitro using the GST pull-down approach and thus lay the foundations for assembly of a recombinant enzyme. All 10 GST-fusion proteins (GST-Pop1p, GST-Pop3p, GST-Pop4p, GST-Pop5p, GST-Pop6p, GST-Pop7p, GST-Pop8p, GST-Rmp1p, GST-Rpp1p and GST-Snm1p), plus GST-alone (to be used as a negative control for the protein–protein interaction studies) were expressed in E. coli and purified by affinity chromatography using glutathione Sepharose (Figure 1A). This is the first report of successful soluble expression and purification all 10 of the yeast RNase MRP subunits, with yields of 15–25 mg protein per 1 l culture. Although the GST-fusion protein is the predominant product in all cases, all lanes show low levels of faster migrating polypeptides that probably represent degradation products or truncations of the recombinant proteins. Less prevalent slower migrating polypeptides are also observed, often common to all preparations, and most likely represent contaminating E. coli proteins. All of the GST-fusion proteins were stable for approximately 1 week when bound to the glutathione Sepharose beads and stored at 48C in the presence of protease inhibitors. Radiolabelling of the GST-fusion protein was highly specific (Figure 1B), with the majority of the radiolabel (60–80%) being incorporated into the GST-fusion protein. Cleavage at the PreScission protease site enabled recovery of radiolabelled protein subunits with the GST-tag removed (Figure 1B). These untagged radiolabelled proteins were then combined with unlabelled GST-fusion protein samples bound to glutathione Sepharose beads to investigate direct one-to-one protein–protein interactions. With the exception of cleaved Snm1p, all RNase MRP protein subunits were stable as both cleaved subunits and as GST-fusions. Cleaved Snm1p was less stable; we were able to use it in protein–RNA interaction screens but were
For the analysis of protein–protein interactions, 50 ml of the beads (washed with PBS and containing bound GSTfusion protein at equivalent loadings, as estimated by SDS-PAGE) were incubated with 50 ml of 1 mM cleaved, untagged radiolabelled protein plus 5 mg BSA for 3 h at room temperature, under continuous agitation. After incubation, the beads were pelleted and the supernatant (containing any unbound cleaved protein) was removed. The beads were then washed three times with 1 ml of PBS + 1% triton + 10 mM DTT and analysed by SDS-PAGE. The gels were vacuum dried to blotting paper, and exposed to PhosphoImager screens. Protein– protein interactions were quantitated using a Typhoon scanner and ImageQuant software. Radiolabelled untagged protein retained on beads bearing a GST fusion protein was quantitated as percentage bound relative to total amount of input radiolabelled protein. In later experiments, NaCl concentration in the final wash was increased to 300 mM or 1 M, from the normal concentration of 150 mM. Filter binding assay For the analysis of RNA–protein interactions, 10 mM of each cleaved, untagged, protein subunit was incubated with 2 nM of annealed, in vitro transcribed 32P-labelled RNA, 1 mM competitor RNA (poly(IC) or poly(U) (Amersham)) and 5 mM MgCl2, on ice, for 20 min in binding buffer (50 mM Tris–HCl, pH7, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Triton-X100). Experiments with poly(IC) and poly(U) were repeated four times in total. Pre-wetted nitrocellulose filter discs (Millipore, 0.45 mm) were placed onto the vacuum filter apparatus, and 0.45 mm filter discs placed on top of these. Both filters were pre-washed with 1 ml of binding buffer, before the sample was pipetted on to the top filter and vacuum applied. Filters were washed with 1 ml of 1 PBS then scintillation counted and the data analysed using Excel. The top 0.45 mm filters are used to remove
RESULTS Preparation of soluble protein subunits of S. cerevisiae RNase MRP
GST-Snm1
GST-Rpp1
GST-Rmp1
GST-Pop8
GST-Pop7
GST-Pop6
GST-Pop5
GST-Pop4
GST-Pop3
GST-Pop1
150 kDa
Rpp1
Snm1
*
+
+
+
−
+/−
+
−
+/−
+
−
3
4
5
6
7
8
9
10
11
12 GST-Snm1
− 2
GST-Rpp1
B
1
GST-Rmp1
Rmp1
Pop8
Pop7
Pop6
Pop5
Pop4
Pop3
Pop1
B
Lane
GST-Pop8
37 kDa
25 kDa
100 1.4 69.3 42.8 100 8.8 26.8 78.4 1.0 33.9 63.3 12.1
interaction
GST-Pop7
% of input
50 kDa
GST-Pop6
*
*
GST-Pop5
*
GST-Pop4
*
GST-Pop3
* *
GST-Pop1
* *
100 kDa
GST-only
75 kDa *
total input
*
37 kDa
25 kDa % of input
100 4.6 100 100 91.6 16.5 100 100 10.8 100 74.1 43.6
interaction
37 kDa
Lane
*
* 25 kDa
GST-only
total input
A
GST-only
GST-Rpp1
GST-Snm1
GST-Rmp1
GST-Pop8
GST-Pop7
GST-Pop6
GST-Pop5
GST-Pop4
GST-Pop3
A
GST-Pop1
6442 Nucleic Acids Research, 2007, Vol. 35, No. 19
*
* *
−
+
+
+
−
+
+
−
+
+
+
2
3
4
5
6
7
8
9
10
11
12
*
* *
1
*
Figure 1. Preparation of RNase MRP protein subunits. (A) Expressed GST-fusion proteins bound to glutathione Sepharose 4B beads. The expression and purity of GST-fusion protein preparations were determined by SDS-PAGE analysis and Coomassie staining. The asterisks () indicate the full-length GST-(fusion) proteins. The bands seen beneath the full-length GST-(fusion) proteins probably represent truncated versions of the full-length recombinant proteins. The sizes of the molecular weight markers are shown on the right. (B) Radiolabelled, cleaved proteins. Whilst bound to glutathione Sepharose, GST fusions were treated with bovine heart kinase in the presence of g-32P-ATP to achieve radiolabelling, followed by removal of the GST-tag by overnight cleavage with PreScission protease. The efficiency of radiolabelling and the purity of the cleaved proteins were assessed by SDS-PAGE analysis followed by exposure to PhosphoImager screens and analysis using a Typhoon scanner. The asterisks () indicate the radiolabelled, untagged proteins. The sizes of the molecular weight markers are shown on the left.
unable to use it to study protein–protein interactions, due to the prolonged incubation times and possible exposure to proteases. Protein–protein interactions Interactions between the bound GST-fusion proteins and the radiolabelled, untagged proteins were analysed by SDS-PAGE and exposure to PhosphoImager screens. Protein–protein interactions were quantitated, relative to the input radiolabelled protein, using a Typhoon scanner and ImageQuantTM software (GE Healthcare). GST-only was included as a negative control, in order to assess the specificity of the interactions. Interactions were assigned as being strong (‘+’; at least 40% of input radiolabelled protein bound to the GST-fusion protein), weak (‘+/’; between 20 and 40% of input protein co-precipitated), or none (‘’;
